Phosphorodiamidate-linked morpholino oligomers, or PMO, are nucleic acid analogs which bind tightly and sequence specifically to complementary RNA and are useful in modulating protein synthesis and thus gene expression. These oligomers are composed of base-pairing recognition moieties (heterocyclic bases) supported by a morpholino backbone system. Morpholino subunits for use in synthesizing such oligomers can be prepared easily from the corresponding ribonucleosides, which are readily available and inexpensive precursors (see e.g. Summerton and Weller, 1993, 1997).
The morpholino nitrogen of a morpholino subunit is typically protected with a trityl or substituted trityl species. During oligomer synthesis, this group must be removed during each cycle to allow incorporation of the next subunit. Failure to completely remove the protecting group leads to N−1 deletion sequences that contaminate the desired oligomer product.
Trityl groups are conventionally removed with acid, and deprotecting reagents used for PMO synthesis have traditionally been carboxylic acids (Summerton et al. 1993, 1997). However, phosphorodiamidate groups are also sensitive to acid, and carboxylic acids useful for detritylation are also capable of promoting hydrolysis of phosphorodiamidate linkages to amidate species, as shown in FIG. 1, with the possibility of more extensive backbone degradation. For example, cyanoacetic acid in 20% acetonitrile/DCM is an effective deprotecting reagent, but it is found to cause substantial (5-10%) hydrolysis of phosphorodiamidate linkages in the PMO product.
Carboxylic acids must also be completely removed from the synthesis support resin prior to the coupling reaction; otherwise, by-products are formed that consist of truncated oligomers containing a 3′-acylated species.
For these reasons, improved reagents are needed for morpholino nitrogen deprotection in PMO synthesis.